Zinc serves as a cofactor in various metallo enzymes and is involved in a broad range of biological processes, such as neurotransmission, gene expression, apoptosis, and immune system functions. Much of the intracellular zinc is bound to proteins and the concentration of free zinc in the cytoplasm is extremely low; however, a fraction of the total zinc is localized in cytoplasmic vesicles and freely exchangeable. There is strong evidence, that these vesicles play a central role in the intracellular regulation of Zn(ll) and possibly other vital biological processes. However, the nature of these processes and the biological function of the vesicles are elusive. The investigation of the dynamics of vesicular zinc has been hampered in particular by the lack of Zn(ll)-specific fluorescent probes, that are suitable for live cell studies. This proposal describes the design, synthesis and characterization of Zn(ll)-specific fluorescent probes, that should overcome problems of existing probes and enable researchers to elucidate the mechanisms of cellular zinc homeostasis. The proposed sensors are specifically designed for two-photon excitation microscopy, a noninvasive live cell imaging technique that drastically reduces photoxicity. The sensing mechanism is based on zinc-induced inhibition of excited state proton transfer (ESIPT), a new design concept that provides large spectral shifts suitable for quantitative emission ratiometric measurements. The proposed research fills a critical niche in the biological chemistry of metal ion speciation. The lack of a non-invasive zinc specific fluorescent sensor is currently a limiting factor in the study of intracellular zinc regulation and trafficking.